KR20210099553A - Injectable sustained-release antibiotics - Google Patents

Abstract

Provided herein are injectable antibiotic compositions for veterinary use. The composition is characterized in that it forms into a gel at the physiological temperature of the animal, wherein the gel is characterized by a stable and reproducible release profile of the antibiotic. A cosolvent has been added to the composition, preferably a cellulose derivative that is at least partially soluble in an organic solvent, and the composition contains a large loading of drug in a poloxamer solution. Methods of treating veterinary infections are also provided.

Description

Injectable sustained-release antibiotics

The present invention relates to a sustained-release preparation, more particularly, to a sustained-release preparation suitable for poorly soluble veterinary antibiotics.

Oral administration of medicaments considered the preferred route in medicine is often not practiced in veterinary medicine, particularly veterinary medicine involving large livestock, for obvious reasons. For similar reasons, administration of a medicament, which must proceed in multiple doses, often proves difficult or even impractical.

In veterinary medicine, sustained release of drugs following parenteral administration is generally preferred over oral administration and enables the treatment of large livestock (eg cattle) as well as companion animals and other animals. Reducing dosing frequency is known to improve patient safety, reduce injection site complications, and improve compliance with drug protocols. Sustained-release formulations alleviate the bolus effect upon injection, exerting a savior-like effect on the side effects caused by the drug. Single or irregular dosing has become the standard procedure for any prophylactic use and treatment. For example, monthly dosing is useful for most heartworm prophylaxis, such as medicaments such as Heartguard®, Sentinel® and Interceptor®. Controlled release parenteral formulations can take the form of liquids, in situ forming solids and solids (Medlicott et al., Advanced Drug Delivery Reviews 2004, 56:1345-1365). The best-selling parenteral controlled-release products are Posilac® breast milk booster (liquid suspension), Micotil® antibiotic (liquid solution), Nuflor® antibiotic (liquid solution) and Contains Revalor® growth promoter (solid implant).

In recent years, studies involving the use of poloxamers in sustained release formulations have been reported. Poloxamers are nonionic triblock copolymers in which blocks of relatively hydrophilic poly(ethylene oxide) (PEO) and relatively hydrophobic poly(propylene oxide) (PPO) are arranged in an ABA triblock structure, that is, PEO-PPO-PEO. am. Poloxamer aqueous gels are described, for example, in US Pat. No. 3,740,421. Poloxamers are used as emulsifiers for intravenous fat emulsions, solubilizers for maintaining the transparency of elixirs and syrups, and wetting agents for antibacterial agents. Poloxamers may also be used in ointment or suppository bases, as tablet binders or coatings [Sweetman (Ed.), Martindale: The Complete Drug Reference, London: Pharmaceutical Press]. The hydrophobic-lipophilic balance (HLB) of a poloxamer can be characterized by the number of ethylene oxide units and propylene oxide units in the copolymer. Due to their amphipathic properties, poloxamer copolymers exhibit surface activity, such as the ability to interact with hydrophobic surfaces and biofilms. This copolymer self-assembles into micelles at a concentration above the Critical Micelle Concentration (CMC) in aqueous solution. The diameter of poloxamer micelles usually varies from approximately 10 nm to 100 nm. The core of micelles consists of hydrophobic PPO blocks isolated from the aqueous exterior by a hydrated shell of PEO blocks. The core may incorporate a variety of therapeutic or diagnostic reagents (Bartrakova & Kabanov, Journal of Controlled Release 2008, 130:98-106). Poloxamers are generally named "letter P (referring to "poloxamer") - three numbers. Multiplying the first two numbers by 100 gives the approximate molecular mass of the PPO core, and multiplying the last number by 10 gives the PEO percentage. For example, P407 is a poloxamer with a PPO molecular mass of 4,000 Da and a PEO content of 70%. According to a further nomenclature (used for example in connection with trade names such as Pluronic® and Lutrol®), copolymers are named with letters that define their physical form at room temperature, "L" for liquid, "P" for paste, "F" for flake (solid), followed by two or three numbers followed by a letter. Multiplying the first number (or the first two numbers in a three-digit number) by 300 gives the approximate molecular weight of the hydrophobic block, and multiplying the last number by 10 gives the percentage of polyethylene oxide (PEO). For example, L61 is a liquid poloxamer with a PPO molecular mass of 1,800 Da and a PEO content of 10% (which will be named P181 according to the naming scheme described above).

US 20090214685 discloses a thermoplastic pharmaceutical composition comprising a botulinum toxin and a biocompatible poloxamer. This pharmaceutical composition, once administered as a liquid, gels after administration into a sustained release drug delivery system from which the botulinum toxin can be released over a period of several days. U.S. Pat. No. 7,008,628 describes pharmaceutical compositions comprising a linear block copolymer, such as a poloxamer terminally modified with a biobinding polymer, such as polyacrylic acid. This polymer can agglomerate as the temperature increases. U.S. Pat. No. 7,250,177 describes gel forming poloxamers modified with crosslinking groups such as acrylates that can be crosslinked to form thermosensitive and lipophilic gels useful for drug delivery or tissue coating. Additional background art includes US Pat. No. 5,035,891 and US Patent Application No. 2004/0247672. International patent application WO 2012131678 (some of the inventors) relates to a sustained release formulation comprising a poloxamer, in the form of a suspension or other form of an insoluble active agent, wherein the formulation disclosed herein comprises an acceptable volume of the administered dose ( While maintaining acceptable volume), it allows the active agent to be used in larger amounts in a single dose.

Florfenicol is a broad-spectrum antibiotic used particularly in the treatment of swine respiratory disease (SRD). Approved veterinary products of florfenicol include injectable formulations, typically containing as much as 300 mg/ml. One of the above injectable products thus approved for veterinary use is dissolved in the organic solvent N-methylpyrrolidone (NMP). Some sustained-release formulations of florfenicol have already been disclosed (eg, Chinese patent application CN103202802 for sustained-release formulations comprising poloxamer and polysaccharide). This invention relates to varying the loading of several different polysaccharides in the formulation and the active agent florfenicol. Pharmacokinetic studies of in vivo gels for controlled delivery of florfenicol in pigs are described in Geng et. al., J. vet. Pharmacol. Therap. 38, 596-600, which demonstrates that the half-life of florfenicol in animal plasma is increased when a poloxamer-based 20% loading gel and a cellulose-based polysaccharide are administered.

There is a need in the art to provide injectable formulations of antibiotics that are capable of releasing the drug in a controlled manner over an extended time interval. There is also a need in the art to provide such agents that will successfully maintain minimal inhibitory concentration levels against a variety of veterinary pathogens. There is also a need in the art to provide antibiotic formulations with high drug loading levels, eg, greater than 25% to about 50%, yet still injectable by conventional syringes.

The stability of sustained release formulations, and the effect that stability has on the release profile of the active agent in the target organism, is in many cases an important factor that has proven to be a delicate balance between the different ingredients in the formulation. Surprisingly, when a combination of a poloxamer and a cellulose derivative at least partially dissolved in an organic solvent, and optionally at least partially dissolved in an organic solvent, is used in a sustained release formulation of an antimicrobial agent, consistent and reproducible release both in vitro and in vivo. It was confirmed that a stable injectable dispersion formulation exhibiting a profile was prepared. Thus, in one aspect, the present invention provides an injectable composition comprising a poorly soluble antimicrobial agent, at least one poloxamer, an organic solvent, a cellulose derivative at least partially soluble in an organic solvent, and an aqueous medium do. Surprisingly, when the active substance exhibits very high loadings (eg, greater than 35 wt % or 40 wt %), the combination in water of a poloxamer with an organic solvent may be sufficient to provide an injectable formulation with a consistent and reproducible release profile. It was also confirmed that Therefore, in a further aspect, the present invention provides an injectable composition comprising an antimicrobial agent, at least one poloxamer, an organic solvent and an aqueous medium, wherein the concentration of the antimicrobial agent is greater than 35 wt% to greater than 40 wt% do.

It therefore comprises a biologically active agent, a poloxamer, an aqueous carrier and an organic cosolvent, injectable at room temperature, wherein the concentration of the active material is 35 wt % or less, and further comprising a cellulosic-based material that is at least partially soluble in an organic solvent. Provided herein are pharmaceutical compositions comprising In one embodiment, if the concentration of drug is greater than 35 wt %, for example 35 wt % to 50 wt % or less than 55 wt %, a cellulosic based material is included. In further embodiments, the concentration of drug is greater than 35 wt%, for example 35 wt% to 50 wt% or up to 55 wt%, such as 40 wt% to 50 wt%, or 42.5 wt% to 50 wt% , or from 45 wt % to 50 wt %, the composition is free of cellulosic based materials. A bioactive agent, a poloxamer, an aqueous carrier, an organic cosolvent, and a cellulose-based material that is at least partially soluble in an organic solvent, wherein the agent is injectable at room temperature, wherein the concentration of the bioactive agent is greater than 10 wt %; Also provided herein are pharmaceutical compositions up to 35 wt %. Bioactive agents include florfenicol, lincomycin, tylosine, metronidazole, tilmicosin, spiramicin, erythromycin, tulasromycin, thiamulin, ampicillin, amoxicillin, clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide, sulfamethoxazole, fluromutilin, avilosine, tilvarosine, doxycycline and oxytetracycline. Preferably the bioactive agent is florfenicol. Also preferably, florfenicol may be present in the composition in a loading amount of from about 25 wt % to about 50 wt %. The organic cosolvent may be present in an amount from about 5 wt % to about 15 wt %. The cellulosic based material that is at least partially soluble in the organic solvent may be hydroxypropyl cellulose. The organic solvent may be selected from the group consisting of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), PEG 400, propylene glycol and ethanol. Preferably the organic solvent is N-methylpyrrolidone. In some preferred embodiments, the pharmaceutical composition comprises the organic solvent N-methylpyrrolidone, the cellulosic material hydroxypropyl cellulose at least partially soluble in the organic solvent, and the bioactive agent florfenicol (concentration of 25 wt % to 50 wt %). %) is included. In some other preferred embodiments, the pharmaceutical composition comprises an organic solvent that is N-methylpyrrolidone and florfenicol in a concentration of 25 wt % to 50 wt %. Also provided herein is a pharmaceutical composition as defined herein for use in treating a veterinary infection in a non-human animal by administering to a non-human animal a pharmacologically effective dose of an antibiotic in said composition. Preferably the composition is administered to said non-human animal once during the course of treatment. Also preferably, administration includes intramuscular injection or subcutaneous injection. In some embodiments, the infection may be caused by a porcine pathogen.

1 schematically shows the florfenicol release profile from selected compositions.
Figure 2 schematically shows the release profile of florfenicol as an influence of the added organic solvent.
Fig. 3 shows the plasma concentration of florfenicol when the composition according to the present invention is administered once versus twice the commercial product.
Figure 4 shows the plasma concentration of florfenicol in one dose of a further composition according to the invention versus two doses of a commercial product.

As described above, the sustained release composition of the present invention comprises an active biological agent. In some embodiments, the biologic is an antimicrobial agent, preferably exhibiting low solubility in aqueous media. Small solubility may be understood, for example, as defined in the existing United States Pharmacopeia, but may be better understood through the context of the formulations described in greater detail below. In a related embodiment, the antimicrobial agent used in the sustained release composition of the present invention is florfenicol, lincomycin, tylosine, metronidazole, tilmicosine, spiramicin, erythromycin, tulasromycin, thiamulin, ampicillin, amoxicillin , clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide, sulfamethoxazole, fluromutilin, avilosine, tilvarosine, doxycycline and oxytetracycline. In some presently preferred embodiments, the antimicrobial agent is florfenicol.

According to the principles of the present invention, the loading (ie the amount of biologically active or antimicrobial agent incorporated into the injectable dosage form) is large, allowing extended and controlled release over several days. Among other factors, many loadings of the injectable compositions of the present invention are facilitated by including in the formulation a biologically active agent, which may be in an insoluble form, to form a dispersion in an aqueous medium. According to the principles of the present invention, the antimicrobial agent dispersed in the formulation is to some extent in the form of a solid. Preferably, more than 90% of the drug has an insoluble form, and 99.999% of the drug may have an insoluble form. Insoluble forms of the drug usually include basic compounds, or specifically salts with less water solubility (although more soluble salts may be known).

The loading may vary depending on the solid phase properties of the active agent. If the drug readily interacts with the aqueous medium, or with the poloxamer or other surfactant, the drug is not easily aspirated into the paste, i.e., into a syringe when the loading value is high (no syringeability and/or ) to form non-injectable compositions. In this case, although such drugs can be used at rather small loading values, such as 12 wt % to 20 wt %, it is generally preferred that the drug loading is large. Thus, in some embodiments, the loading is at least about 20 wt % of the injectable composition.

In some other embodiments, the loading is from about 25 wt % to about 30 wt % of the injectable composition. In some other embodiments, the loading is at least about 30 wt % of the injectable composition. In some further embodiments, the loading is from about 30 wt % to about 45 wt % of the injectable composition. In some further embodiments, the loading is from about 35 wt % to about 50 wt % of the injectable composition. In some embodiments, the loading is from about 30 wt % to about 35 wt % of the injectable composition. In some specific embodiments, when the bioactive agent is florfenicol, the florfenicol loading applied in a particular application is 25 wt% to 50 wt%, such as 28 wt% to 32 wt%, or 36 wt% to 42 wt %, or 44 wt % to 48 wt %.

The biologically active agent forms a dispersion with a cosolvent in an aqueous medium. It is understood that the biologically active agent will be in the form of a solid, such as a powder. The powder may be in the form of agglomerates, granules or coated powders, but preferably, the powder is a neat drug ingredient powder having a limited particle size distribution. In some preferred embodiments, the particle size of the powder is less than about 90 microns, more preferably less than about 50 microns. It can also often be advantageous to use a powder having a smaller particle size, or even a micronized powder. While not wishing to be bound by theory, it is believed that the smaller particle size powder may increase the highest plasma concentration achievable from the formulation in vivo compared to ordinary drug powder, but the difference in vitro is unlikely to be small or significant. . Finely divided powders or reduced particle size powders can be prepared, as is generally known in the art, for example by high-impact milling or high-shear milling, sieving under pressure and It can be obtained directly from the biologically active ingredient powder by other methods.

In certain preferred embodiments, the biologically active substance or antimicrobial agent is released from the body-forming gel of the composition of the present invention for at least 3 days. In some other embodiments, the substance is released over two to three days. In some further embodiments, the substance is released over 4 to 5 days. In some embodiments, the agent is released from the single injectable composition of the present invention for more than 5 consecutive days. Release can therefore be described in terms of duration of release rather than in terms of any specific rate. The duration of release in vivo can be identified in plasma as a drug concentration that maintains a significant level over time. In another embodiment, the duration of release in vivo can be identified in a target organ or tissue as a drug concentration that maintains a significant level over time. Specifically, as long as the active agent is an antibiotic, the duration of release can be ascertained in plasma and the obtained concentration can be compared with the minimum inhibitory concentration of the antibiotic for a particular pathogen. Since sink conditions are maintained in vitro, for example, the duration of drug release under conditions as described in the Examples section below can be from about 12 hours to about 3 days.

According to some of the principles of the present invention, an advantageous combination of an organic cosolvent, a poloxamer in an aqueous medium, and a cellulose derivative that is at least partially soluble in an organic solvent exerts a synergistic effect and is stable over several days of the bioactive agent. and controlled release is achieved. The drug loading in a formulation comprising such a cellulose derivative may be as low as about 5 wt % or about 10 wt %. However, depending on the solid phase nature of the antibiotic, the drug loading may be as high as 35 wt%, or 40 wt%, or 45 wt%, or 47.5 wt%, or even 50 wt%. Moreover, it was unexpectedly found that relatively stable and repeatable drug release kinetics can be achieved from a composition comprising a poloxamer as defined herein, water and an organic cosolvent when the active agent is present in a concentration greater than 35 wt %. became While the presence of cellulose derivatives that are at least partially soluble in organic solvents has been shown to be advantageous even at high drug loadings, the release profile in the absence of excipients surprisingly meets the requirements for drug release variability of existing US Pharmacopoeia controlled release dosage forms. consistent enough to satisfy However, when the drug loading is less than or equal to 35 wt %, it is preferred that the composition comprises a cellulose derivative as described below.

According to some embodiments, the poloxamer as described above is selected from the group consisting of poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, and combinations thereof. In some presently preferred embodiments, the poloxamer as described above is poloxamer 407.

Since the presence of a poloxamer allows the composition to gel under physiological temperatures, the poloxamer must be present in the injectable composition at a concentration suitable to form a stable gel, specifically when large amounts of undissolved active agent powder are present. do. Therefore, the concentration of poloxamer as described above exceeds 8% by weight of the total weight of the formulation. Depending on the properties of the drug, such as particle size, solubility of the drug, affinity of the drug for the poloxamer, and the loading of the drug, the amount of poloxamer ranges from as little as 7 wt% to 9 wt% to 16 wt% to 20 wt%. can be up to

The synergistic effect of some of the embodiments of the present invention is achieved by combining the poloxamer with a unique combination of an organic cosolvent and a cellulose derivative that is at least partially soluble in the organic solvent. The chemical compatibility between the cellulose derivative and the organic solvent, and the ratio between these two components, together with the poloxamer concentration, determines the release profile of the bioactive agent. Without wishing to be bound by any mechanism or theory, when an organic solvent can increase the solubility of a biologically active agent, it also slows down the release of the active agent from the gel-like composition under physiological conditions due to its effect on the gel itself. is hypothesized. It is also hypothesized that, for at least some drugs, the addition of cellulose derivatives as described above may be involved in increasing the release rate of the bioactive agent, and that organic solvents contribute to reducing the variability over time of the overall release profile. Although the molecular weight of the cellulose derivative to be used may be selected according to the release profile considered and the rheological properties required according to some embodiments of the present invention, the concentration ratio between the cellulose derivative and the organic solvent is usually about 1:6 to about 1:20. If the drug is present in particularly high loadings, such as from greater than 35 wt % to greater than 40 wt %, the concentration ratio between the cellulose derivative and the organic solvent may be from about 1:10 to about 1:100.

Cellulose derivatives that are at least partially soluble in organic solvents are usually appreciably soluble to some extent in conventional pharmaceutical organic solvents, such as ethanol. Preferably suitable derivatives produce clear solutions when dissolved, for example, by 1 gram in 100 mL of 96% ethanol at room temperature. One suitable as a cellulose derivative that is at least partially soluble in an organic solvent is hydroxypropylcellulose. Hydroxypropylcellulose has the additional useful property of being very soluble in aqueous solution at room temperature, but becoming less soluble with increasing temperature. Without being bound by any theory or mechanism of action, when the composition of the present invention is injected into an animal, the solubility of hydroxypropylcellulose decreases, which contributes to the stability of the formed gel, thereby contributing to the release of the bioactive agent. It is hypothesized that better control will be achieved.

In some related embodiments, the concentration of the cellulose derivative as described above is from about 0.5 wt % to about 1.5 wt % of the total weight of the injectable composition. In some other embodiments, the cellulose derivative concentration is from about 0.5 wt % to about 1 wt %. When the drug is present at very high loading levels (eg, greater than 40%), the concentration of the cellulose derivative can be from about 0.05 wt% to about 0.7 wt%.

In some embodiments, the organic solvent as described above is selected from the group consisting of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), PEG 400, propylene glycol and ethanol. In some presently preferred embodiments, the organic solvent is NMP.

In some related embodiments, the concentration of the organic solvent as described above is from about 1.5 wt % to about 20 wt % of the total weight of the injectable composition. In some other embodiments, the concentration of the organic solvent is from about 3 wt % to about 15 wt %. In some other embodiments, the concentration of the organic solvent is from about 8 wt % to about 12 wt %.

In further related embodiments, the at least one poloxamer, the organic solvent and the cellulose derivative are dissolved in the aqueous medium. The aqueous medium is usually water, optionally water comprising further dissolved additives such as salts and/or buffers. In general, the amount of aqueous medium in the formulation is the remainder after deducting each of the percentages of the biologically active agent, at least one poloxamer, cellulose derivative, cosolvent, and other excipients, if used, from 100% of the composition. The salt may include sodium chloride, calcium chloride or magnesium chloride, and the buffer may include monobasic, dibasic or tribasic salts of alkali metals and phosphates.

The synergistic effect that can be exerted on cosolvents in aqueous media, cellulose derivatives at least partially soluble in organic solvents, and poloxamers when the drug is present in very high loadings, such as greater than 35 wt % to greater than 40 wt %, is While clearly advantageous, the effect of cellulose derivatives on system stabilization reduces the need for obtaining pharmaceutically acceptable compositions that exhibit a release profile with a relative standard deviation of the concentration values at each time point (eg 10% or less). can As demonstrated in the examples below, for example, when hydroxypropylcellulose is excluded from a florfenicol formulation with a loading of 47.5 wt %, there is a slight burst effect accompanied by an increase in the relative standard deviation (RSD) at the initial time point. However, an acceptable release profile was also achieved.

According to the principles of the present invention, the formulation achieved is a stable and injectable formulation at room temperature (eg 15° C. to 25° C.) or low temperature (eg 2° C. to 8° C.) for animals (eg body temperature greater than 35° C.). It is a formulation characterized by a reproducible and well-controlled release profile of an incorporated bioactive agent that transforms into a gel when injected into the body of

In another aspect, the present invention provides a method for preparing an injectable sustained release formulation comprising an antimicrobial agent in an aqueous medium, at least one poloxamer, an organic solvent, and a cellulose derivative at least partially dissolved in the organic solvent; 1) mixing water and an organic solvent (known as a co-solvent), preferably cooling the resulting mixture, 2) adding at least one poloxamer and the cellulose derivative to the [cooling] mixture of step 1 added sequentially or concomitantly, followed by mixing until dissolved, and 3) adding the antimicrobial agent to the resulting mixture.

In some embodiments, the organic solvent as described above is selected from the group consisting of N-methylpyrrolidone (NMP), DMSO, PEG 400, propylene glycol and ethanol. In some presently preferred embodiments, the organic solvent is NMP.

In some embodiments, the poloxamer as described above is selected from the group consisting of poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, and combinations thereof. In some presently preferred embodiments, the poloxamer as described above is poloxamer 407.

In some embodiments, the cellulose derivative is hydroxypropylcellulose.

In some embodiments, the antimicrobial agent used in step 3 is florfenicol, lincomycin, tylosine, metronidazole, tilmicosin, spiramicin, erythromycin, tulathromycin, thiamulin, ampicillin, amoxicillin, clavulan acid, penicillin, streptomycin, trimethoprim, sulfonamide, sulfamethoxazole, fluromutilin, avilosine, tilvarosine, doxycycline and oxytetracycline. In some presently preferred embodiments, the antimicrobial agent is florfenicol.

As used herein and in the claims, the term "biologically active agent" is interchangeable with "antibacterial agent", "drug" or "antibiotic".

As used herein and in the claims, the term "cosolvent" refers to an organic solvent that is miscible with the aqueous carrier or water in the formulations of the present invention. In some embodiments, the organic solvent as described above is selected from the group consisting of N-methylpyrrolidone (NMP), DMSO, PEG 400, propylene glycol and ethanol.

In a further aspect, an injectable sustained release composition injection as generally described herein is administered to a morbid individual in need of treatment for a veterinary infection, comprising an antimicrobial agent in an aqueous medium, at least one poloxamer, an organic solvent, and optionally Provided are methods of treating a veterinary infection by administering at least one injection of a composition comprising a cellulose derivative at least partially dissolved in an organic solvent, and use of the composition in the treatment of a veterinary infection. Preferably, the method comprises a single administration of the agent, but more than one injection dose may be used as needed and depending on the length of the treatment period. When dealing with veterinary diseased individuals, it is advantageous to minimize touching, in order to reduce the stress on the animal and the effort required to find, capture, and touch the sick animal. Therefore, it is preferable to finish the administration at one time. Alternatively, the method comprises administering the agent multiple times, so long as the number of administrations is less than the number of times currently required for the particular biologically active agent.

If the injection volume is to be large, administration may include a single injection, or multiple injections into multiple sites. When a poorly soluble drug is present in a sufficient amount in a relatively small injection volume, the advantages of the formulations of the present invention may eliminate the need to apply multiple injection sites.

Administration is usually intramuscular injection. However, the administration may also be subcutaneous administration, intraperitoneal administration, intradermal administration, or may be special site administration, such as intravaginal administration in cattle and sheep, intracaudal administration or otic administration in beef cattle, intramammary administration, and the like.

Veterinary infections that can be treated according to the present invention include infections caused by swine pathogens, infections of cattle, infections of poultry, infections of companion animals, or infections of animals and wild animals in zoos.

In some embodiments, the organic solvent as described above is selected from the group consisting of N-methylpyrrolidone (NMP), DMSO, PEG 400, propylene glycol and ethanol. In some presently preferred embodiments, the organic solvent is NMP. In some embodiments, the poloxamer as described above is selected from the group consisting of poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, and combinations thereof. In some presently preferred embodiments, the poloxamer as described above is poloxamer 407. In some embodiments, the cellulose derivative is hydroxypropylcellulose. In some embodiments, the antimicrobial agent is florfenicol, lincomycin, tylosine, metronidazole, tilmicosine, spiramicin, erythromycin, tulathromycin, thiamulin, ampicillin, amoxicillin, clavulanic acid, penicillin, strepto is selected from the group consisting of mycin, trimethoprim, sulfonamide, sulfamethoxazole, fluromutilin, avilosine, tilvarosine, doxycycline and oxytetracycline. In some presently preferred embodiments, the antimicrobial agent is florfenicol.

Example

Materials and Methods

Florphenicol and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich (Israel). Poloxamers 407, 188, 338 and 237 were obtained from BASF's local responsible company. Amoxicillin, tylosine, Klucel® polymer (hydroxypropylcellulose), PEG400 and propylene glycol were donated from pharmaceutical companies. Water was purified and distilled on a column before use. Sodium chloride was purchased from Merck (Israel).

Unless otherwise specified, florfenicol injectable formulations were prepared as follows:

Measured amounts of water and cosolvent were mixed at room temperature and then dissolved by adding salt and buffer if present in the formulation and mixing. Weighed amounts of poloxamer and cellulose derivative were cooled to 4° C. in a cooling chamber; Separately, the mixture of water and cosolvent was also cooled. Then, under the same conditions, the polymer was added to the mixture of water and cosolvent, followed by vigorous mixing using a magnetic stirrer until a clear solution was obtained. Thereafter, florfenicol powder (flakes) was added to the solution obtained therefrom, and then mixed for 24 hours in a cooling room to ensure excellent distribution in the formulation. Alternatively, especially for high loading formulations, a metered amount of florfenicol is placed in a mortar and made into a geometrically homogeneous mixture, i.e., a metered aliquot of the preparation solution is used. Approximately equal aliquots of solution were mixed in a mortar until exhausted.

gel point measurement

While increasing the temperature, the glass tube containing 0.5 mL to 1 mL of the formulation was turned upside down to check whether it was gelled. The temperature at which the formulation no longer flows down when the glass tube is inverted was regarded as the primary gel point. Alternatively, the temperature was raised to 40° C. for preliminary screening and the time taken for the formulation to form a gel was recorded.

The gel point was also measured rheologically using the Anton Paar Rheometer Physica MCR 101, i.e. a parallel plate spindle isolated with a 200 μm gap (temperature sweeping at a shear rate of 100 reciprocal seconds). did. The second derivative of the viscosity curve gave the temperature at which the viscosity changes most rapidly, which was considered the true gelation point.

Florphenicol confirmed

Florfenicol was confirmed via HPLC (HP1090 instrument, using UV detector measuring absorbance at 224 nm). A C-18 250x4.6 5 μm column was used, with an elution rate of 1.2 ml/min and an ACN:DDW mobile phase ratio of 25:75. Florfenicol was eluted from 4 min to 4.5 min under these conditions.

dissolution test

To test the dissolution rate of florfenicol from the formulation, a syringe barrel of a 5 mL syringe was cut into 2 mL sections and used as a tube-shaped holder. The injectability of the formulation was evaluated by sealing one side with a Parafilm® sheet, accurately weighing approximately 2 mL of an aliquot of the formulation at room temperature, and placing it in the tube holder prepared above by using a suitable syringe (19G needle). . The top side was then sealed with another Parafilm® sheet and placed in a preheated oven (40° C.) for at least 15 minutes to ensure gelation. The Parafilm® sheet was then securely removed and transferred directly to the Caleva 6ST Dissolution Tester (USP Apparatus 2) (set at 20 rpm and 40°C) with the tube holder placed in a sinker basket. The temperature was selected to match or similar to the body temperature of the target animal (pig). The dissolution medium was phosphate buffer USP, pH 6.8, and a volume of 900 mL per tube holder was applied. A sample was taken from the dissolution medium at the scheduled time, and then the missing volume was filled with fresh dissolution medium. At the end of the test, the tube holder was washed in the dissolution vessel and then vigorously mixed to prepare an amount of material recovered to be used as a 100% standard. The percentile of the highest florfenicol concentration at each time point was reported with standard deviation.

Additionally, dissolution of some of the florfenicol compositions was performed using USP Apparatus 5 (“paddle over disk”) as specified below.

Example 1 - Comparative Example

A. In order to evaluate the efficacy of the formulations disclosed in Chinese patent application CN103202802, Example 7 (florfenicol 30%) of the above publication was reproduced and tested under the conditions described. Since this publication contains only a very brief guide on the grade of hypromellose used, two grades (HPMC K4M and HPMC K15M) having an apparent viscosity of 20 cP or less under the test conditions of low concentration were tested separately. Briefly, the poloxamer was accurately weighed, cooled, dissolved in a large amount of cold water (4° C.), and then hydroxypropylmethylcellulose (HPMC) was added thereto. After receiving the remainder of the excipient from the stock solution, water was added to make up the remainder, followed by thorough mixing. After preparing a formulation sample having a total weight of 25 grams, a sample prepared using HPMC K4M was named Sample Formulation 1.1, and a sample prepared using HPMC K15M was named Sample Formulation 1.2.

To test the advantageous effect of the organic solvent according to the invention, the same formulations as described above were prepared, but this time using N-methylpyrrolidone as a cosolvent in an amount of about 20 wt % of the total weight of the solvent (water 20 % is replaced by organic solvent), thereby preparing sample formulation 1.3 and sample formulation 1.4 corresponding to HPMC K4M and HPMC K15M, respectively.

It was confirmed that the sample prepared according to 1.1 and the sample prepared according to 1.2 at room temperature could not be sucked into the syringe without a needle. This is to show that the formulation prepared according to the invention of CN103202802 (Example 7) appeared to be non-injectable under the reported conditions. In order to obtain the release profile and results of the non-injectable formulations, a spatula was used to hold the sample. Addition of NMP as a cosolvent increased the viscosity to an impractical extent (hard gels were obtained even at 4°C), although the samples prepared according to 1.3 and 1.4 were also uninjectable, even though these samples were not It should be further mentioned that a test for emission was carried out.

B. Formulations with 20% loading were prepared according to the trends of Examples 7 and 6 of Prior Art Publication CN103202802 for the preparation of injectable compositions. In summary, florfenicol loading was reduced with water. Sample formulation 1.5 contained HPMC K15M and pure water, and sample formulation 1.6 contained HPMC K15M and 20 wt% NMP (cosolvent). Formulations corresponding to formulations 1.5 and 1.6 containing 20 wt% of florfenicol were easily injected and gelled through the test needle under the conditions of sample preparation for dissolution testing.

Although the compositions prepared according to 1.1 to 1.4 lacked injectable properties, the compositions were placed into tubes using a spatula in a conventional round semi-solid filling technique to test the florfenicol release profile from the aforementioned formulations. The results are presented in Table 1 below.

For example, when comparing Formulation 1.1 and Formulation 1.3, Formulation 1.2 and Formulation 1.4, and Formulation 1.5 and Formulation 1.6, addition of NMP to a composition comprising HPMC generally accelerated the release rate of florfenicol from the formulation, Sometimes it can be seen in Table 1 that the variability decreased.

Likewise, it can be seen that the injectable formulation according to the prior art publication could only have a florfenicol loading of only 20%, which is also reflected in another publication of the same inventor (ZX Geng, HM Li, J. Tian, TF Liu). , ZG Yu, J Vet Pharmacol Ther, Vol 38, Iss 6, Dec 2015, 596-600). At higher loadings, injectable compositions could not be obtained when using the formulations according to the prior art.

Example 2

In order to evaluate the advantages of the florfenicol sustained-release formulation according to the principle of the present invention by comparing it with another known gel-based sustained-release formulation disclosed in International Patent Application WO2012131678, a gel containing 30% by weight of florfenicol was prepared. . The effects of cosolvent NMP, the cellulose-based material hydroxypropylcellulose, and their synergistic combinations were studied separately. All formulations showed a gelation behavior at 25°C to 35°C (each data is presented below), and the release profile was evaluated according to the above method. The formulations are summarized in the table below along with the release profile data for each of these formulations.

Formulation 2.1 is according to an embodiment of the present invention, comprising both hydroxypropylcellulose, a cellulosic-based material; Formulation 2.2 exhibits a co-solvent exclusion effect; Formulation 2.3 exhibits hydroxypropylcellulose (Klucel® EF) and co-solvent NMP exclusion effect; Formulation 2.4 is a comparative preparation according to WO2012131678, which contains neither a cosolvent nor a cellulose derivative. Formulation 2.5 (an embodiment of the invention) showed a lower loading (20 wt % florfenicol) and Formulation 2.6 contained 20% florfenicol but no NMP, compared to Formulation 2.6 it became

When the results of Formulation 2.1 and Formulation 2.3 were compared, it was easily observed that when NMP was added to the formulation according to WO 2012131678, a significant decrease in drug release was caused, but the variability between the results was also significantly reduced. Furthermore, the addition of hydroxypropylcellulose to the formulation according to WO 2012131678 resulted in a significant decrease in the release rate and a relatively large variability in the release profile. The results showed that only the addition of both components (NMP and HPC) produced a synergistic effect, resulting in less variability (smaller standard deviation of the mean compared to the mean), and the purely aqueous formulation 2.2 and 2.3, the drug release amount was also increased. Moreover, the formulation according to the invention, ie the formulation with a loading level of 20%, exhibited approximately the same or slightly greater decrease in florfenicol release, with the proviso that the variability was CN' containing hypromellose instead of hydroxypropylcellulose. It could be confirmed that the variability of the hypothetical 20% formulation of 802 (formulation 1.5) was smaller than that of the 802.

The results are summarized in Table 2 below and also in FIG. 1 . The release profile is presented in Figure 1 with error bars representing the RSD at each time point. Diamonds (♦) represent formulation 2.1, solid squares (■) represent formulation 2.2, solid triangles (▲) represent formulation 2.3, and an x (x) represents formulation 2.4, “% FFC” indicates Florpe It represents the cumulative release percentile of nicol, and "t(h)" represents the elapsed time (hours) from the start of the experiment.

Example 3

To evaluate the effect of the selected cosolvent NMP on the formulation, a gel was prepared from formulation 2.1, but the NMP content was varied from 5 wt% to 20 wt% to prepare formulations 3.1 (5 wt%) and 3.2 (20 wt%). provided.

The release data is presented in Table 3 below, and the profile is presented in FIG. 2 with error bars representing the RSD at each time point. Diamond (♦) represents formulation 3.1 (labeled “5 wt%”), solid squares (■) represent formulation 2.1 (designated “10 wt%”), and solid triangles (▲) represent formulation 3.2 (“20 wt%"), "% FFC" represents the cumulative release percentile of florfenicol, and "t(h)" represents the elapsed time (hours) from the start of the experiment.

When NMP was 5 wt%, the variability increased, while the emission profile remained almost unchanged, and it was confirmed that the emission was slightly accelerated at only 20%.

Example 4

To evaluate the effect of additional cosolvents on the formulation, a gel was prepared from formulation 2.1, but NMP was either DMSO (Formulation 4.1), propylene glycol (Formulation 4.2), PEG 400 (Formulation 4.3) or ethanol (Formulation 4.4). replaced.

The release profiles are summarized in Table 4 below.

It could be readily seen that both DMSO and PEG 400 gave almost the same release profile as when NMP was used, but significantly more and significantly reduced the gel point of the solution.

Example 5

To demonstrate the in vivo effect of the present invention, a pharmacokinetic study was performed to demonstrate that the plasma levels were maintained and effective for a single dose of florfenicol to pigs. This study was approved by the Ethics Committee for Animal Research studie of the Hebrew University in Jerusalem. A total of 6 animals including 2 female pigs aged 3 to 4 months were used. A 20G central venous catheter was inserted into each porcine jugular vein to facilitate blood collection. In the first arm of the study, all animals were administered 40 mg/kg of Formulation 2.1 as a one-shot treatment followed by Nuflor® (Merck Animal Health - Florphenicol 30% solution in NMP) at 48 hour intervals. Different test treatments were performed in the second arm, either administered twice at 20 mg/kg, or after a wash out period of 2 weeks.

Each treatment dose wjs (0 hour), and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours, 30 hours, 52 hours, 72 hours, 96 hours, 144 hours after the first dose and blood samples were drawn at 196 hours. After collecting the sample and putting it in a heparinized tube, plasma was immediately separated, and then stored at -20°C until analysis. On the day of analysis, samples were spiked with an internal standard (chloramphenicol) and then extracted with acetonitrile. The standards were established on the same day. Parent drug florfenicol and its main metabolite using UHPLC-MS/MS (positive ion mode TSQ Quantum Access Max mass spectrometer, multi-response monitoring (MRM) mode and electrospray ionization (ESI) applied for duplicate acquisitions) Florphenicol-amine was identified. Results were obtained for florfenicol (parent compound) and florphenicol-amine (main metabolite).

Data analysis was performed using Microsoft Excel software. The area under the curve (AUC) was obtained by numerical integration. After confirming the final slope by inverse transformation, the slope was calculated by fitting the curve to the exponential decrease data. All further calculations were performed using the fitted function. No deconvolution was performed due to the complexity of the model, specifically for the double injection arm. For this Nuflor® arm, the final slope data was also used to extrapolate the 48 hour time point. Data were calculated from the mean curve; Where applicable, ranges of individual values are provided.

For appropriate comparison, the results of a plot of plasma concentration versus time, prepared for the parent florfenicol compound, are presented in FIG. 3 . The dotted line in each graph represents the potential highest MIC90 for common porcine respiratory disease target pathogens. Error bars represent standard error of the mean. Arrows indicate dosing times. Diamonds (♦) indicate Nuflor ("Treatment: Nuflor 20 mg/kg x 2, n=2"), and solid squares (■) indicate Formulation 2.1 ("Treatment: P2.1, 40 mg/kg"). x 1, n=5”), “concentration (μg/ml)” indicates the plasma concentration of florfenicol, and “time (hour)” indicates the time (hour) elapsed from the start of the experiment indicates.

The pharmacokinetic parameters obtained for this data are summarized in Table 5 below.

The elimination half-life of Nuflor® at the second injection was calculated; The data at the first injection showed a significantly shorter half-life, indicating a rapid dissipation in the early stages. The highest concentration reported for Newflor cancer is the highest concentration at the first injection.

As evidenced by the time percentiles for the MIC and the AUIC, it was readily confirmed that the formulation according to the present invention produced a higher exposure associated with florfenicol compared to the marketed product when injected once.

Example 6

In order to evaluate the ability of the system to handle extremely high drug loadings, the following florfenicol formulations were also prepared according to the procedures described herein. Formulation 6.1 contained about 33 wt% florfenicol, Formulation 6.2 contained about 36 wt% florfenicol, and Formulation 6.3 contained about 39 wt% florfenicol.

The composition could be injected through a 16G needle and exhibited the reverse thermal behavior after enabling injection, such as gelling on heating and liquefying again on cooling. The release profile and rheological data are summarized in Table 6 below.

As evidenced by the low relative standard deviation at each time point, it could be readily confirmed that the formulations formed a gel with increasing temperature and released the drug in a controlled fashion with little variability.

A further composition with a loading of 45 wt % or greater was prepared. Florfenicol was sieved through a 50 micron mesh to obtain a lower particle size fraction. Formulations and results are summarized in Table 7 below.

Dissolution tests were performed using the "paddle over disk" method. An amount of about 1 g was tested in 900 mL of USP phosphate buffer (pH 6.8) with the addition of 1% CTAB. Rheological measurements were performed with a gap of 500 μm at 500 inverse seconds.

From the results, it can be seen that the smaller particle size did not adversely affect the release profile, and that when the loading was very high, it probably accelerated the drug release, even slightly, and that the extremely high loading of florfenicol could be achieved as an injectable formulation. could confirm that there was

Example 7

An additional composition with a loading of 47.5 wt % was prepared. As in Example 6, sieved florfenicol was used. The formulations and results are summarized in Table 8 below.

From the results, it could be easily confirmed that a composition comprising 47.5% by weight of florfenicol, for example, exhibits excellent viscosity at a suitable gel point in air, and can be prepared for injection.

In addition, it was confirmed that even when hydroxypropylcellulose was small (see, e.g., Formulation 7.1 vs. Formulation 6.7), the release profile remained stable, but the RSD was relatively small (although in fact the variability of 7.1 was slightly greater). .

Unexpectedly, the variability in the absence of hydroxypropylcellulose (formulation 7.2) was still within the pharmaceutically acceptable range, but even when the cellulose additive was in an amount of only 0.1%, the variability was significantly reduced, provided that The release profile was not adversely affected. Moreover, the addition of more cosolvents (Formulation 7.3 vs. Formulation 7.1) reduced the variability even more, and even more for Formulation 7.2, which contained no cellulose additive.

Example 8

To further demonstrate the in vivo effect of the present invention, a pharmacokinetic study was conducted to demonstrate that a single dose of florfenicol to pigs is maintained and efficacious with prolonged plasma levels.

According to the manufacturer's recommendations, 40 mg/kg of formulations 6.6 to 6.8 were simultaneously treated in one shot for a total of 20 pigs, or Nuflor® (Merck Animal Health - Florphenicol 30% solution in NMP) 30 mg/kg kg was administered. Also, a formulation comprising 40 wt% of florfenicol, 12 wt% of poloxamer 407, 0.5 wt% of Klucel EF, 5 wt% of NMP and 42.5 wt% of water (designated herein as 8.1) (gel point = 21.7°C) ) was administered at 40 mg/kg. The release profile of Formulation 8.1 under the same conditions as in Example 7 is shown in Table 9 below.

0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 50 hours, 72 hours, 84 hours, 96 hours, 120 hours , blood samples were taken at time points 144 and 168 hours.

A plot of florfenicol plasma concentration versus time is presented in FIG. 4 . Fig. 4 shows the plasma concentrations of florfenicol at each sample collection time. A diamond (♦) indicates Newflor, a solid square (■) indicates formulation 8.1, a solid triangle (▲) indicates formulation 6.6, an x mark (x) indicates formulation 6.7, and an asterisk (*) indicates formulation 6.8 , "C(ng/mL)" represents the plasma concentration of florfenicol, and "t(h)" represents the elapsed time (hours) from the start of the experiment.

From the results, it was readily confirmed that the commercial product was rapidly dissipated from pig blood, whereas all of the formulations according to the present invention maintained plasma levels above 1000 ng/mL for an average of 72 to 84 hours. It is noteworthy that the dose-corrected AUC for treatment was approximately the same between groups, indicating that bioavailability was not reduced by the controlled release formulation. Peak plasma concentrations were clearly the highest in the immediate release commercial product; Formulation 6.7 showed a significantly higher peak concentration than Formulation 6.8, which only differed in the particle size of the drug.

Streptococcus suis ( virulent pathogen in pigs) over time (currently considered to be 2 mcg/mL) of the minimum inhibitory concentration and the time of exceeding the hourly minimum inhibitory concentration of the tested products are presented in Table 10 below.

From the results, it was clear that the test formulations according to the present invention showed superior results in rescuing S. suis with significant clinical potential.

Example 9

In order to demonstrate the ability of the composition according to the present invention to release other antibiotics, a formulation comprising 30 wt% of amoxicillin was prepared. Formulation 9.1 contained both a cosolvent and a cellulose derivative (hydroxypropylcellulose) that was at least partially soluble in an organic solvent, Formulation 9.2 contained only hydroxypropylcellulose, Formulation 9.3 contained any of the additional excipients did not contain any. Formulations were prepared according to the procedure as described for florfenicol.

The composition could be injected through a 16G needle and exhibited the reverse thermal behavior after enabling injection, such as gelling on heating and liquefying again on cooling. The release profile data are summarized in Table 11 below.

As evidenced by the low RSD at each time point, the formulations formed a gel and released the drug in a controlled fashion with little variability, but late drug release when neither NMP nor Klucel was present. It was easy to see that it became more inconsistent, which may indicate that a less stable gel was formed in the absence of both excipients.

Example 10

To further demonstrate the ability of the composition according to the present invention to release other antibiotics, formulations comprising 15 wt % of tyrosine were prepared. Formulation 10.1 contained both a cosolvent and a cellulose derivative (hydroxypropylcellulose) that was at least partially soluble in an organic solvent, Formulation 10.2 contained only hydroxypropylcellulose, Formulation 10.3 contained any of the additional excipients did not contain any. Formulations were prepared following the procedure as described for florfenicol.

The composition could be injected through a 16G needle and exhibited the reverse thermal behavior after enabling injection, such as gelling on heating and liquefying again on cooling. The release profile data are summarized in Table 12 below.

The formulation was formed into a gel and it could be readily confirmed that it released the drug in a controlled fashion. Formulation 10.1 was made to contain slightly more poloxamer to compensate for the increased drug dissolution due to NMP. 10.1 showed a low variability release profile as evidenced by the low RSD at each time point, especially at the intermediate time point. Formulation 10.2 showed slightly greater variability, except that drug release became more variable when neither NMP nor Klucel were present.

Claims (18)

A bioactive agent, a poloxamer, an aqueous carrier and an organic cosolvent, wherein the cellulosic-based material is injectable at room temperature and is at least partially soluble in an organic solvent, provided that the concentration of the active agent is 35 wt% The following pharmaceutical compositions. The pharmaceutical composition according to claim 1, wherein the concentration of the biologically active agent is 10 wt% to 35 wt%. The pharmaceutical composition of claim 1 , wherein the concentration of the biologically active agent is greater than 35 wt % and the composition is free of cellulosic-based substances that are at least partially soluble in an organic solvent. The pharmaceutical composition of claim 1 , wherein the concentration of the biologically active agent is greater than 35 wt %, and wherein the composition further comprises a cellulosic based material that is at least partially soluble in an organic solvent. 5. The pharmaceutical composition according to claim 3 or 4, wherein the concentration of the biologically active agent is 35 wt% to 50 wt%. 6. The method of any one of claims 1 to 5, wherein the bioactive agent is florfenicol, lincomycin, tylosine, metronidazole, tilmicosine, spiramicin, erythromycin, tulasromycin, thiamulin, ampicillin, A pharmaceutical composition selected from amoxicillin, clavulanic acid, penicillin, streptomycin, trimethoprim, sulfonamide, sulfamethoxazole, fluromutilin, avilosine, tilvarosine, doxycycline and oxytetracycline. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the biologically active agent is florfenicol. 7. The pharmaceutical composition of claim 6, wherein said biologically active agent is present in said composition in a loading amount of about 25 wt% to about 50 wt%. 9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the organic cosolvent is present in an amount from about 5 wt% to about 15 wt%. 10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the cellulosic-based material at least partially soluble in the organic solvent is hydroxypropylcellulose. The pharmaceutical according to any one of claims 1 to 10, wherein the organic solvent is selected from the group consisting of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), PEG 400, propylene glycol and ethanol. composition. 12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the organic solvent is N-methylpyrrolidone. 13. The bioactive agent according to any one of claims 1 to 12, wherein the organic solvent is N-methylpyrrolidone, the cellulosic-based material at least partially soluble in the organic solvent is hydroxypropylcellulose, and the bioactive agent is florfenicol at a concentration of 25 wt% to 50 wt%. 14. The method according to any one of claims 1 to 13, wherein the organic solvent is N-methylpyrrolidone, the bioactive agent is florfenicol, and the concentration of florfenicol is between 35 wt% and 50 wt%. % of the pharmaceutical composition. 15. Use according to any one of claims 1 to 14 for treating a veterinary infection in a non-human animal by administering to the animal a pharmacologically effective amount of an antibiotic in a pharmaceutical composition for use in treating said veterinary infection. A pharmaceutical composition for The pharmaceutical composition according to claim 15, wherein the composition is administered to the non-human animal once per treatment course. The pharmaceutical composition according to claim 15 or 16, wherein said administration comprises intramuscular injection or subcutaneous injection. 18. The pharmaceutical composition according to any one of claims 15 to 17, wherein said infection is caused by a porcine pathogen.

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